Identification of truncated variants in GLI family zinc finger 3 (GLI3) associated with polydactyly

Background Polydactyly is a prevalent congenital anomaly with an incidence of 2.14 per 1000 live births in China. GLI family zinc finger 3 (GLI3) is a classical causative gene of polydactyly, and serves as a pivotal transcription factor in the hedgehog signaling pathway, regulating the development of the anterior-posterior axis in limbs. Methods Three pedigrees of polydactyly patients were enrolled from Hunan Province, China. Pathogenic variants were identified by whole-exome sequencing (WES) and Sanger sequencing. Results Three variants in GLI3 were identified in three unrelated families, including a novel deletion variant (c.1372del, p.Thr458GlnfsTer44), a novel insertion-deletion (indel) variant (c.1967_1968delinsAA, p.Ser656Ter), and a nonsense variant (c.2374 C > T, p.Arg792Ter). These variants were present exclusively in patients but not in healthy individuals. Conclusions We identified three pathogenic GLI3 variants in polydactyly patients, broadening the genetic spectrum of GLI3 and contributing significantly to genetic counseling and diagnosis for polydactyly. Supplementary Information The online version contains supplementary material available at 10.1186/s13018-024-04928-0.


Ethical compliance
This study was approved (2,023,030,444) by the Ethics Committee of Xiangya Hospital, Central South University, Changsha, China.We performed this study in accordance with the principles outlined in the Declaration of Helsinki.The patients/participants or their guardians provided written informed consent to participate in the study.

Participants/patients
Three families (Family I-III) were investigated in this study.Peripheral blood samples were collected from probands and their family members.Clinical data were recorded carefully.

Whole-exome sequencing
Genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA).Exome capture and whole-exome sequencing (WES) were conducted at Berry Genomics (Beijing, China). 1 µg DNA was captured using the SureSelect Human All Exon Kit V6 (Agilent Technologies, Inc., CA, USA) and sequenced using the Illumina HiSeq4000 platform (Illumina Inc., CA, USA).Briefly, the genomic DNA was randomly extracted using a Covaris S220 sonicator (Covaris, Inc., MA, USA).The fragmented DNA underwent three enzymatic steps: end repair, a-tailing, and adapter ligation.The adapterligated DNA fragments were amplified using Herculase II Fusion DNA Polymerase (Agilent).Finally, the exosomes in the pre-capture libraries were captured using the Sure-Select capture library kit (Agilent).After DNA quality assessment, the captured DNA library was subjected to WES on the Illumina HiSeq4000 platform.Downstream processing was carried out using the Genome Analysis Toolkit (GATK), Varscan2, and Picard, and variant calls were made with the GATK Haplotype Caller 12. Variant annotation was performed according to Ensemble release 82, and filtering was conducted using ANNOVAR Documentation.

Co-segregation analysis
Co-segregation analysis was performed on each family member using Sanger sequencing.The primer pairs (GLI3-1-F: ) used for PCR amplification were designed using Primer 5.The sequences of the PCR products were determined using an ABI 3100 Genetic Analyzer (ABI, Foster City, CA, USA) [22].

Clinical description
We conducted an investigation into three Chinese families (Family I-III) from Hunan Province of China (Table 1).Family I consists of six immediate members, four of whom have exhibited features of polydactyly or syndactyly (Fig. 1A).Radiography showed that the proband presented syndactyly of the fourth and fifth fingers on the right hand and polydactyly of thumbs on each foot (Fig. 1B).Additionally, his sister was affected by syndactyly on all limbs, and his mother had surgically corrected dysmorphic toes.The family reported a history of polydactyly in the deceased grandfather's toes.
Proband II from Family II was born with polydactyly affecting both hands and feet, with no similar conditions reported in the family's history (Fig. 1C and D).Surgical removal of the extra fingers was performed at a different hospital when the patient was one year old, and subsequent toe resection occurred at our hospital, resulting in successful recover (Fig. 1E).
Proband III from Family III displayed heptadactyly on both feet, polydactyly on the first and fifth toes, and syndactyly between the first and second toes, with no reported familial history of these anomalies (Fig. 1F and  G).The extra toes were surgically removed (Fig. 1H).Considering the clinical manifestations in Family I and  III, we made primary diagnosis of mild GCPS, whereas the proband from Family II appeared to have IPD [13].

Genetic analysis
Using WES and Sanger sequencing, we identified three GLI3 variants in the probands from three families, including two novel variants (NM_000168.6:c.1372del, p.Thr458GlnfsTer44 and c.1967_1968delinsAA, p.Ser656Ter) and one known variant (NM_000168.6:c.2374 C > T, p.Arg792Ter).According to the standards of the American College of Medical Genetics and Genomics (ACMG) standards, these variants were designated as "pathogenic" (Table 2).
In Family I, WES produced 9.7 Gb of data, achieving 98.1% coverage of the target region and over 10× coverage for 99.0% of the targets.After excluding common variants in the 1000G and GnomAD databases, and retaining variants predicted to be disease-causing using Muta-tionTaster, Polyphen-2, SIFT and CADD, which were also positioned in genes associated with bone development, one GLI3 variant (c.1372del, p.Thr458GlnfsTer44) was identified.Sanger sequencing further confirmed the heterozygous frameshift variant in GLI3 in Proband I (Fig. 2A).Adherence to ACMG guidelines, this variant was deemed "pathogenic": it was a frameshift variant often leading to loss of function (PVS1), was absent in control cohorts from the 1000G and GnomAD databases (PM2) and co-segregated with the polydactyly phenotype in Family I (PP1).It was also predicted to have a deleterious effect on the gene product as determined by tools such as MutationTaster (PP3).Additionally, alignments of GLI3 amino acid sequences across various species showed high conservation in this region (p.458-466) (Fig. 2B), demonstrating the critical nature of this sequence.
In Family II, we identified the heterozygous nonsense variant (c.1967_1968delinsAA, p.Ser656Ter) in Proband II (Fig. 2C).According to the ACMG guidelines, this variant was classified as a nonsense variant (PVS1).It has not been observed in healthy population databases (PM2), and bioinformatic tools predict its deleterious effects (PP3).Consequently, it was designated as "pathogenic".
It has been suggested that the location of GLI3 variants significantly impacts the manifestation of digital anomalies due to the dual role of GLI3 as an activator or repressor in the SHH pathway [29].In mouse model, GLI3 haploinsufficiency, often resulting from loss-of-function variants upstream of or within the ZFN domain, has been linked to the pathogenesis of GCPS, characterized by cranial enlargement and a wide interorbital distance [30].In this study, the frameshift variant p.Thr458GlnfsTer44 in Family I introduces a premature stop codon, leading to a GLI3 protein lacking the essential ZFN domain for DNA binding, which may contribute to GCPS, corroborating earlier research findings.The CS domain is critical in regulating the ratio of GLI3-A/GLI3-R, thus, variants in the CS region can result in an excess of GLI3-R in limb buds and the neural tube, which predominantly drives the pathogenesis of PHS.PHS is associated with diverse anomalies including hypothalamic hamartoma, cleft larynx, imperforate anus, and pulmonary lobation anomalies [31].This study also identified two truncation variants (p.Ser656Ter and p.Arg792Ter) within the CS domain, which may result in premature termination of GLI3, loss of GLI3-A function, and excessive production of GLI3-R (Fig. 2E).Notably, while the patient in Family II was diagnosed with IPD, and the patient in Family III with mild GCPS, neither presented craniofacial anomalies.Research by Sczakiel et al. indicated that GLI3 variants linked to IPD can also occur in the ZFN and CS domains, challenging previous beliefs that such variants are confined to the C-terminal TAD domain, aligning whit our findings in Family II [32].
A previous study proposed that GLI3 expression could demarcate between posterior and anterior hand anomalies.Specifically, GLI3 haploinsufficiency has been linked to the etiology of preaxial polydactyly, whereas postaxial polydactyly arises from abnormal truncations in the TAD [33].In this study, Proband I, exhibited preaxial polydactyly traits, carried a truncating variant upstream of the ZFN in GLI3, likely leading to GLI3 haploinsufficiency.Conversely, Proband II, who displayed postaxial polydactyly, harbored a truncating variant in the CS domain of GLI3, potentially resulting in TAD functional deficiency.These observations align with findings from Bass et al.However, despite the variant in Proband III being associated with a TAD deficiency, the phenotype included both preaxial and postaxial polydactyly, highlighting gaps in current understanding of the relationship between GLI3 variant locations and clinical manifestations.Future research is warranted to elucidate this association more comprehensively.
To investigate the genotype-phenotype correlation further, we analyzed 246 cases of polydactyly linked to GLI3 variants (Fig. 3A).A predilection for variants in the C-terminal region of GLI3 was noted.Statistical analyses confirmed that the majority of polydactyly cases resulted from by loss-of-function GLI3 variants (Fig. 3B), with 144 cases (approximately 59%) associated with GCPS (Fig. 3C), corroborating previous studies [13].In this study, both Family I and III demonstrated features of GCPS.Furthermore, prior research estimated the incidence of IPD at 1.01 per 1000 live births [34], and our analysis revealed that a only 13% of IPD cases involved truncating GLI3 variants (Fig. 3D).In this study, all patients exhibited GLI3 truncations, yet the patients in Family II was diagnosed with IPD, whereas patients in Family I and III showed milder forms of GCPS, primarily affecting hands or feet.These findings suggest a tendency for milder phenotypic manifestations among GLI3 truncation carriers in Hunan Province.Remarkably, a further statistical assessment of all IPD patients confirmed that loss-offunction GLI3 variants are also a predominant pathogenic factor (Fig. 3E).
In this study, surgical procedures were performed on patients affected by polydactyly to remove the supernumerary fingers.This process may result in damage to the muscles and bones in the affected areas, impacting the patients' daily lives during the recovery period.Recent studies have identified multiple genes associated with susceptibility to skeletal muscle injury and repair capacity [35][36][37][38][39][40], and significant differences in single nucleotide polymorphisms (SNPs) of these genes have been observed among populations with different levels of physical activity [41,42].These findings suggest that analyzing specific SNPs of susceptibility genes in patients could be beneficial in predicting their postoperative recovery capacity.For patients with high muscle susceptibility and low repair capacity, it may be advisable to consider therapeutic measures that promote muscle repair during surgery.For example, based on the ability of mesenchymal stem cells (MSCs) to differentiate into various mesenchymal tissues, applying MSCs to the injury site may significantly improve the biomechanical properties, structure, and function of the muscle postoperatively [43].

Fig. 1
Fig. 1 Clinic description of the probands with polydactyly related to GLI3.(A) Pedigree of Family I. (B) Clinical features of proband I. (C) Pedigree of Family II.(D) Clinical features of proband II before surgery.(E) Clinical features of proband II after surgery.(F) Pedigree of Family III.(G) Clinical features of proband III before surgery.(H) Clinical features of proband III after surgery.Squares = men; circles = women; black symbols = individuals with variants; Slashes = individuals who die; arrows = the probands

Fig. 2
Fig. 2 Genetic description of the probands with polydactyly related to GLI3.(A, C, D) Sanger sequencing results of the GLI3 variant among the probands.(B) Alignment analysis of the region (p.458-466) in the GLI3 amino acid sequence showed that the region was highly conserved.(E) Localization of the variant in GLI3.Red region = frameshift variant; RD = repressor domain; SUFU = SUFU site; ZFN = zinc finger domain; CS = cleavage site, TAD = transactivation domain

Table 1
Clinical details of patients in this study Bil, bilateral; 1, thumb or toe; 2/4/5, the second/fourth/fifth finger

Table 2
Information and pathogenicity classification of GLI3 variants in this study D, disease causing; AD, autosomal recessive